CAN YOUR HPLC COLUMN DO THIS ?
Type B SILICA
TYPE-C SILICA 
A Reverse Phase Chromatography Comparison of
Unbonded Type B and TYPE-C Silica™ HPLC Columns
Using an Aqueous Mobile Phase
Uracil & Phenol
Phenol
Uracil
Biphenyl
Toluene
Toluene
Column: 75 x 4.6mm id
Isocratic:
60% - 0.04% v/v H3PO4
40%ACN
Biphenyl
Type B Silica
Flow rate: 1ml/mn
TYPE-C Silica™
Cogent UDC-Cholesterol
on TYPE-C Silica™
Cogent UDC-Cholesterol
on TYPE-C Silica™
Neutral Compounds
Cogent HPS C18
Cogent UDC-Cholesterol
Flow Rate: 3ml/min
Wavelength: 254nm
Solvents
A: Buffer
B: 100%ACN
Acidic Compounds
Cogent HPS C18
Cogent UDC-Cholesterol
Flow Rate: 3ml/min
Wavelength: 254nm
Solvents
A: Buffer
B: 100%ACN
Cogent UDC-Cholesterol
on TYPE-C Silica™
Phenol & Pyridine
Peak Order
Neutral
Peak Order
Acid
Peak Order
pH 8.00
pH 2.18
pH 2.18
5% 20mM NH4COOH/NH4OH
95% Acetonitrile
60% (H3PO4)
40% Acetonitrile
90% (H3PO4)
10% Acetonitrile
Phenol-Pyridine Compounds
Cogent HPS C18
Cogent UDC-Cholesterol
Flow Rate: 3ml/min
Wavelength: 254nm
Solvents
A: Buffer
B: 100%ACN
Basic Compounds
Cogent HPS C18
Cogent UDC-Cholesterol
Flow Rate: 3ml/min
Wavelength: 254nm
Solvents
A: Buffer
B: 100%ACN
Cogent UDC-Cholesterol
on TYPE-C Silica™
Normal Phase Resolution of Basic Antidepressants
Solvents:
A: 10%
B: 90%
Solvents:
A: 8%
B: 92%
Solvent A: 20mM NH4 COOH/NH4OH
Solvent B: Acetonitrile
pH 8.00
RETENTION TIME MAP UDC-CHOLESTEROL
Showing Both Normal and Reverse Phase Utility
Column Dimensions 150x4.6mm
REAL LIFE, UNIQUE APPLICATION:
METFORMIN
GLYBURIDE
THE CHALLENGE:
Separate: LogP of –2.64 & +4.79… Isocratically
COGENT UDC-CHOLESTEROL w/ TYPE-C SILICA
A:
B:
99.95% Aqueous - 0.05% H3PO4(pH 2.18)
99.95% Acetonitrile - 0.05% H3PO4
Flow Rate:
Column:
UV :
Back pressure:
1 ml/min
75 x 4.6mm id
210nm
600 psi
Metformin log P = -2.64
Glyburide log P = +4.79
Same Column – ISOCRATIC RUNS
By changing solvent composition, the
elution order can be reversed
I AQUEOUS NORMAL PHASE: NON POLAR FIRST!
II AQUEOUS REVERSE PHASE: POLAR FIRST!
If 2 min. analysis with baseline resolution is too
slow…WHY NOT INCREASE FLOW RATE?
Let us review…
TYPE- C Silica™unbonded
compared to
Cogent-UDC Cholesterol™
Aqueous Reverse Phase & Organic Normal Phase of
Carvone on TYPE-C Silica™- Unbonded
AQUEOUS Reverse Phase = MeOH
A: 80%
B: 20%
AQUEOUS Reverse Phase = THF
A: 90%
D: 10%
AQUEOUS Reverse Phase = ACN
A: 90%
E: 10%
A: 0.05%v/v H3PO4 (pH 2.15
B: MeOH
C: Acetonitrile
D: THF
E: Hexane
F: Ethyl Acetate
ORGANIC Normal Phase = Hexane & Ethyl Acetate
E: 95%
F: 5%
Flow Rate:1ml/min
Column: 75 x 4.6mm id
UV: 255nm
TYPE-C SILICA™ CAN SEPARATE
METFORMIN FROM GLYBURIDE
METFORMIN
73% 0.05% v/v H3PO4
27% Acetonitrile
Flow:
1ml/mn
Column: 75 x 4.6mm id
UV:
255nm
Metformin
Glyburide
HOWEVER…
COGENT UDC-CHOLESTEROL w/ TYPE-C SILICA
A: 99.95%
B: 99.95%
Aqueous - 0.05% H3PO4(pH 2.18)
Acetonitrile - 0.05% H3PO4
Flow Rate:
Column:
UV :
Back pressure:
1 ml/min
75 x 4.6mm id
210nm
600 psi
Metformin log P = -2.64
Glyburide log P = +4.79
Same Column – ISOCRATIC RUNS
By changing solvent composition, the
elution order can be reversed
I AQUEOUS NORMAL PHASE: NON POLAR FIRST!
II AQUEOUS REVERSE PHASE: POLAR FIRST!
MIXED SEPARATION MECHANISMS FOR DIFFERENT
COMPOUNDS
The Aqueous Normal Phase Separations of Metformin on both bonded phase
Cogent UDC-Cholesterol™ and TYPE-C Silica™-unbonded Columns are the
same.
This suggests that the silica surface of both columns provides the main
separation mechanism for Metformin regardless of the bonded phase.
BUT…
Compare this data to the Aqueous Reverse Phase separations of Glyburide.
The bonded Cogent UDC-Cholesterol™ column, under the same conditions,
separates Glyburide much differently from that of the Type-C Silica™unbonded column.
This suggests that the bonded phase of the UDC-Cholesterol™ column
provided the separation mechanism for Glyburide but not Metformin, under
these conditions.
HOW DOES THIS WORK WITH METFORMIN?
The Aqueous Normal Phase Separations of Metformin at approximately
50% acetonitrile results in a longer retention time as the acetonitrile percentage
increases. This occurs at the same percentage for both the un-bonded and
bonded phases.
This suggests that the silica surface of both columns provides the main
separation mechanism for Metformin regardless of the bonded phase.
HOW DOES THIS WORK WITH GLYBURIDE?
The Aqueous Reverse Phase separation of Glyburide on the TYPE-C
Silica™-unbonded column shows retention at 27% acetonitrile, but no
retention at 35%. With the bonded UDC-Cholesterol™ column, even at 80%
acetonitrile, the Glyburide is retained.
The bonded Cogent UDC- Cholesterol™ column, under the same
conditions, separates Glyburide much differently from that of the TYPE-C
Silica™ - unbonded column.
This suggests that the bonded phase of the UDC-Cholesterol™ column
provided the separation mechanism for Glyburide but not Metformin under
these conditions.
COGENT UDC-CHOLESTEROL™
SEPARATION MECHANISM FOR
GLYBURIDE
It can be seen that the silica hydride has no reverse
phase retention above 35% acetonitrile.
The reverse phase retention on the
Cogent UDC-Cholesterol™ column above 50%
acetonitrile must, therefore, be totally a function of
the bonded phase.
This suggests that the retention characteristics of
the bonded phase must be taken into account
when considering the retention by reverse phase,
as well as, the compound itself.
KEY DIFFERENCES OF
TYPE-C™ PHASES
A key issue to understanding the uniqueness of TYPE-C™
phases is that it is possible to have different compounds
(Metformin and Glyburide) use different separation
mechanisms (ANP & ARP) in the same analysis on the same
column.
Within the mechanisms above, the choice of TYPE-C
Silica™-unbonded, bonded Cogent UDC-Cholesterol™ or
bonded Cogent Bidentate C18™, can each produce a
reverse phase response; which can be due to the silica
hydride surface or the bonded phase as dominant factors in
the separation or combinations of the two.
SELECTIVITY COMPARISON IN AQUEOUS REVERSE
PHASE OF NEUTRALS ON BONDED AND UN-BONDED
PHASES
Cogent UDC-Cholesterol™
bonded
1
2
Method Conditions
3
40% 0.05% v/v H3PO4
60% Acetonitrile
Flow:
1ml/mn
Column: 75 x 4.6mm id
UV:
255nm
4
TYPE-C Silica™
un-bonded
Method Conditions
1
2
3
4
60% 0.05% v/v H3PO4
40% Acetonitrile
Flow:
1ml/mn
Column: 75 x 4.6mm id
UV:
255nm
SELECTIVITY COMPARISON IN AQUEOUS REVERSE
PHASE OF URACIL, PYRINDINE, PHENOL
Cogent UDC-Cholesterol™
bonded
1
2
Method Conditions
80% 0.05% v/v H3PO4
20% Acetonitrile
Flow:
1ml/mn
Column: 75 x 4.6mm id
UV:
255nm
3
TYPE-C Silica™
un-bonded
Method Conditions
1
2
3
90% 0.05% v/v H3PO4
10% Acetonitrile
Flow:
1ml/mn
Column: 75 x 4.6mm id
UV:
255nm
SELECTIVITY COMPARISON IN AQUEOUS
REVERSE PHASE OF AROMATIC ORGANIC ACIDS
Method Conditions
80% 0.05% v/v H3PO4
20% Acetonitrile
Cogent UDC-Cholesterol™
bonded
Flow:
1ml/mn
Column: 75 x 4.6mm id
UV:
255nm
1
2
3
TYPE-C Silica™
un-bonded
Method Conditions
90% 0.05% v/v H3PO4
10% Acetonitrile
1
2
3
Flow:
1ml/mn
Column: 75 x 4.6mm id
UV:
255nm
WHAT ABOUT
PHASE COLLAPSE
WITH
100% AQUEOUS ON
TYPE-C™ PRODUCTS?
USING 100 % AQUEOUS ON
COGENT UDC-CHOLESTEROL™ WITH TYPE-C SILICA™
3
After 3 hours of
100% aqueous
exposure, results
are the same as
with the first
injection
7
2
1
4
8
6
5
1- OXALIC ACID
2- TARTARIC ACID
3- FORMIC ACID
4- ACETIC ACID
5- SODIUM AZIDE
6- URACIL
7- FUMARIC ACID
8- PROPIONIC ACID
100% 0.05% v/v H3PO4
Flow Rate: 1ml/mn
Column:
75 x 4.6mm id
UV:
255
USING 100 % AQUEOUS ON pH STABLE
COGENT BIDENTATE C18™ WITH TYPE-C SILICA™
1
After 3 hours of
100% aqueous
exposure,
results are the
6 same as with
the first
injection
4
1- OXALIC ACID
2- FORMIC ACID
3- ACETIC ACID
4- SODIUM AZIDE
5- URACIL
6- FUMARIC ACID
7- PROPIONIC ACID
2 3
5
7
100% Water w/0.05% v/v H3PO4
Flow Rate:1ml/mn
Column: 75 x 4.6mm id
UV:
255
WATER SOLUBLE VITAMINS ON
COGENT BIDENTATE C18™ WITH TYPE-C SILICA ™
Method Conditions
0-4 min:
95% 0.1% v/v TFA in DW
5% Acetonitrile
4-10 min:
85% 0.1% v/v TFA in DW
15% Acetonitrile
10-15 min:
Equilibrate start conditions
Flow Rate: 1ml/min
Column:
250 x 4.6mm id
Cogent Bidentate C18
UV:
270nm
Peaks
Vitamin
1- Niacinamide
B3
2- Thiamine Mono-Nitrate B1
3- Pyridoxine HCl
B6
4- Folic Acid
B11
5- Riboflavin
B2
LogP
- 0.45
- 3.43
- 0.56
- 2.81
- 1.05
WHAT ABOUT
PEPTIDES?
PEPTIDE ANALYSIS ON COGENT UDC-CHOLESTEROL™
RT: 3.6 min
30%-Distilled Water
70% -Methanol
Peptide 6
11 Amino Acid Residues
Centered around Aspargine
Flow Rate: 0.5ml/mn
Column: 150 x 4.6mm id
UV:
210nm
RT: 2.4 min
20%-Distilled Water
70% -Methanol
Peptide 9
11 Amino Acid Residues
Centered around Aspargine
PEPTIDE ANALYSIS ON COGENT UDC-CHOLESTEROL™
30%- Distilled Water
RT: 3.7 min.
RT: 2.4 min.
70%- Methanol
10%- Distilled
Water
90%- Methanol
Peptide 11
9 Amino Acid Residues
Centered around Histidine
Flow Rate: 0.5ml/mn
Column: 150 x 4.6mm id
UV:
210nm
Peptide 11
9 Amino Acid Residues
Centered around Histidine
SO WHICH
TYPE-C™ PHASE
SHOULD I CHOOSE?
THE ANSWER IS A
SCOUT KIT ™
Cogent Scout Kits™
150mm x 4.6mm ID
Scout Kit™
75mm x 4.6mm ID
Mini-Scout Kit™
50mm x 3.0mm ID
50mm x 2.1mm ID
20mm x 4.0mm ID
LC-MS Methods Kit™
LC-MS Methods Kit™
Ballistic LC-MS Kit™
20mm x 2.0mm ID
Mini-bore Scout Kit™
Cogent Scout Kits™

Each Mini-Scout Kit™ contains:
5 HPLC Columns
2 Super-Link End Fittings
1 Super-Link Column Coupler
1 Instruction Manual
Why Use 5 Different Phases
in Method Development?
Cogent HPS  C18
Isocratic Separation
1
(1) Phenol
2+4
(2) Dimethyl Phthalate
5
3+6
(3) Toluene
(4) Nortriptyline
(5) Imipramine
(6) Amitryptiline
3mL/min 20mM NH4COOH
& NH4OH pH 8.00
Cogent HPS Cyano
(1) Phenol
Isocratic Separation
(2) Dimethyl Phthalate
1+2+3
(3) Toluene
4+5+6
(4) Nortriptyline
(5) Imipramine
(6) Amitryptiline
3mL/min 20mM NH4COOH
& NH4OH pH 8.00
Multi-Mode Chromatography
Cogent HPS 
C18
Coupler
Cogent HPS
Cyano
Multi-Mode w/ Cogent HPS
C18 and Cyano
(1) Phenol
Isocratic Separation
(2) Dimethyl Phthalate
(1)
(5)
(4)
(6)
(3) Toluene
(2)
(4) Nortriptyline
(3)
(5) Imipramine
(6) Amitryptiline
3mL/min 20mM NH4COOH
& NH4OH pH 8.00
Multi-Mode Chromatography
• Unlike Mixed Mode Chromatography
• Multi-Mode is reproducible…every time
Cogent Scout Kits™ Provide:
Speed
 Selectivity Choices
 Versatility
 Multi-Mode™ Option
 Multiple Column Size Choices for LCMS
